CN116730902B - Method for synthesizing liraglutide - Google Patents

Method for synthesizing liraglutide Download PDF

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CN116730902B
CN116730902B CN202310982723.5A CN202310982723A CN116730902B CN 116730902 B CN116730902 B CN 116730902B CN 202310982723 A CN202310982723 A CN 202310982723A CN 116730902 B CN116730902 B CN 116730902B
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liraglutide
resin
fmoc
synthesizing
main chain
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CN116730902A (en
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王辉平
王佳炜
邢海英
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Hangzhou Peptide Biochem Co ltd
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C07C233/46—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/47—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
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Abstract

The invention discloses a method for synthesizing liraglutide, belonging to the technical field of polypeptide synthesis; the method comprises the following steps: s1: firstly, under the condition of an activating agent, synthesizing Fmoc-Gly-resin by solid phase of the resin and glycine; s2: deprotection of Fmoc-Gly-resin, and sequential coupling according to the amino acid sequence of the liraglutide main chain peptide by adopting a solid phase synthesis method to obtain the liraglutide main chain resin; s3: under alkaline condition, coupling reaction is carried out on lysine of the liraglutide main chain resin and the liraglutide intermediate of the formula I, pH is regulated, cracking is carried out, crude products are obtained, and pure liraglutide is obtained after purification. The liraglutide prepared by the invention has higher yield and purity.

Description

Method for synthesizing liraglutide
Technical Field
The invention belongs to the technical field of polypeptide synthesis, and particularly relates to a method for synthesizing liraglutide.
Background
Liraglutide is a GLP-1 (glucagon-like peptide) analog with 97% sequence homology to human GLP-1, which can bind and activate the GLP-1 receptor. GLP-1 receptor is a target of natural GLP-1, GLP-1 is an endogenous incretin hormone, and can promote the glucose concentration of pancreatic beta cells to dependently secrete insulin.
At present, the existing preparation method of liraglutide mainly comprises the following steps: (1) genetic engineering; the method mainly has the defects of great technical difficulty, high equipment requirement, large-scale production risk, high cost and adverse industrialized production; (2) a stepwise coupled solid phase synthesis method; the problems that it has are: because the liraglutide peptide chain is longer, gradual coupling often results in incomplete coupling, many mismatched peptide impurities and racemized peptide impurities are generated, the yield of crude peptide is low, the impurities are difficult to remove, and great difficulty is caused to the purification of the crude peptide. (3) fragment synthesis; the existing fragment synthesis method has low crude peptide yield and fails to inhibit/reduce the generation of very similar and similar impurities such as mismatched peptide impurities, racemic peptide impurities and the like. Therefore, the synthesis method of the liraglutide with high product yield is provided with important practical significance.
Disclosure of Invention
The invention aims to provide a liraglutide intermediate, which is subjected to coupling reaction with lysine of a liraglutide main chain resin to prepare a liraglutide crude peptide, so that the yield of the liraglutide crude peptide is improved, and the liraglutide with higher yield is obtained.
The technical scheme adopted by the invention for achieving the purpose is as follows:
1. a liraglutide intermediate has a structure shown in formula I:
a formula I;
wherein R is an active intermediate having a structural formula selected from at least one of the following:
。
according to the invention, the liraglutide intermediate is prepared from the active intermediate R to Palmitoyl-Glu-OtBu, possibly has higher grafting activity, can be better grafted on lysine, and can obtain crude liraglutide peptide with higher yield, thereby obtaining pure liraglutide with higher yield and purity.
Still more preferably, the structural formula of the liraglutide intermediate is selected from at least one of the following:
。
the invention also discloses application of the liraglutide intermediate in preparation of liraglutide.
Furthermore, the invention also discloses application of the liraglutide intermediate in improving the yield of the liraglutide.
It is another object of the present invention to provide a method for synthesizing liraglutide, comprising the steps of:
s1: firstly, under the condition of an activating agent, synthesizing Fmoc-Gly-resin by solid phase of the resin and glycine;
s2: deprotection of the Fmoc-Gly-resin, and sequential coupling according to the amino acid sequence of the liraglutide main chain peptide by adopting a solid phase synthesis method to obtain the liraglutide main chain resin;
s3: under alkaline condition, coupling reaction is carried out on lysine of the liraglutide main chain resin and the liraglutide intermediate, pH is regulated, cracking is carried out, crude products are obtained, and pure liraglutide is obtained after purification.
According to the invention, firstly, an active intermediate is grafted to Palmitoyl-Glu-OtBu to prepare a liraglutide intermediate, and then the liraglutide intermediate is subjected to coupling reaction with lysine of a liraglutide main chain resin to prepare a liraglutide crude peptide, so that the yield of the liraglutide crude peptide is improved, the liraglutide intermediate possibly has higher grafting activity and can be better grafted to lysine to obtain the liraglutide crude peptide with higher yield, and then a pure liraglutide with higher yield and purity is obtained.
According to the method for synthesizing liraglutide of the present invention, the active agent is selected from at least one of HOBt, DIC, DIEA, HATU, DIPEA and PyBOP.
According to the method for synthesizing liraglutide of the present invention, the base used under alkaline conditions is at least one selected from triethylamine, N-methylmorpholine, N-diisopropylethylamine, sodium acetate, potassium acetate, sodium phosphate, disodium hydrogen phosphate, potassium phosphate, dipotassium hydrogen phosphate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate and sodium hydroxide.
According to the method for synthesizing liraglutide, the molar ratio of the liraglutide main chain resin to the liraglutide intermediate is 0.05-0.1:1, and particularly preferably 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1 and 0.1:1.
According to the method for synthesizing liraglutide, the temperature of the coupling reaction is between-20 ℃ and 60 ℃; preferably 0-40deg.C, more preferably 0 deg.C, 1 deg.C, 2 deg.C, 3 deg.C, 4 deg.C, 5 deg.C, 6 deg.C, 7 deg.C, 8 deg.C, 9 deg.C, 10 deg.C, 11 deg.C, 12 deg.C, 13 deg.C, 14 deg.C, 15 deg.C, 16 deg.C, 17 deg.C, 18 deg.C, 19 deg.C, 20 deg.C, 22 deg.C, 23 deg.C, 24 deg.C, 25 deg.C, 26 deg.C, 27 deg.C, 28 deg.C, 29 deg.C, 30 deg.C, 31 deg.C, 32 deg.C, 33 deg.C, 34 deg.C, 35 deg.C, 36 deg.C, 37 deg.C, 38 deg.C, 39 deg.C, 40 deg.C.
According to the method for synthesizing liraglutide, the pH range is 5-14; particularly preferably 7 to 12.
According to the invention, firstly, an active intermediate is grafted to Palmitoyl-Glu-OtBu to prepare a liraglutide intermediate, and then the liraglutide intermediate is subjected to coupling reaction with lysine of a liraglutide main chain resin to prepare a liraglutide crude peptide, so that the yield of the liraglutide crude peptide is improved, the liraglutide intermediate possibly has higher grafting activity and can be better grafted to lysine to obtain the liraglutide crude peptide with higher yield, and then a pure liraglutide with higher yield and purity is obtained.
Detailed Description
The invention discloses a solid-phase preparation method for synthesizing liraglutide, which can be realized by appropriately improving process parameters by a person skilled in the art by referring to the content of the invention. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
In the process of synthesizing liraglutide, the experimental methods used are conventional experimental methods unless otherwise specified.
In the synthesis process of liraglutide, the solvent is generally a conventional solvent selected from any one of DCM, THF, ethyl acetate, acetonitrile, DMF, DMSO, NMP, water, acetone, dimethylacetamide and hexamethylphosphoramide; DMF, DMSO, DMAC, HMP, acetonitrile and water systems, acetone and water systems are particularly preferred.
According to the method for synthesizing liraglutide disclosed by the invention, the Resin is selected from at least one of 2-CTC Resin, resin or king Resin.
According to the method for synthesizing liraglutide, the used lysate is a mixture of TFA, phenylsulfide, anisole and EDT, and the volume ratio is 85-90:2-5:1-5:1-5.
According to the method for synthesizing liraglutide, the preparation steps of Fmoc-Gly-resin in S1 are as follows: weighing resin, adding the resin into a solid phase reaction column, washing the resin with a solvent, swelling the resin with the solvent for 25-45min, dissolving Fmoc-Gly-OH in the solvent, adding an activating agent under the ice bath condition for activation, adding the activating agent into the solid phase reaction column for reaction, adding anhydrous methanol for sealing for 1-2h, washing the resin with the solvent for 3-5 times, sealing the resin with the anhydrous methanol for 20-40min, and draining the solvent to obtain the Fmoc-Gly-resin with the substitution degree of 0.1-0.5mmol/g.
Further, in the preparation step of Fmoc-Gly-resin in S1, the substitution degree of the resin is 0.25-1.0mmol/g.
Further, according to the method for synthesizing liraglutide of the present invention, in the preparation step of Fmoc-Gly-resin in S1, the weight ratio of resin, fmoc-Gly-OH and activator is 2-4:1-2:1-3.
According to the method for synthesizing liraglutide, the preparation steps of the liraglutide main chain resin in S2 are as follows: weighing Fmoc-Gly-resin, adding the Fmoc-Gly-resin into a solid phase reaction column, washing for 2-4 times by using a solvent, swelling the Fmoc-Gly-resin by using the solvent for 25-45min, deprotecting, washing to obtain H-Gly-resin, dissolving Fmoc-Arg (Pbf) -OH and an activating agent into the solvent to form a mixed solution, adding the mixed solution into the solid phase reaction column, reacting for 2-4H at room temperature, repeating the deprotection step, adding corresponding amino acid for coupling according to the peptide sequence of the main chain of the liraglutide, fmoc-Gly-OH, fmoc-Arg (Pbf) -OH, fmoc-Val-OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Lys- (Alloc) -OH, fmoc-Ala-OH, fmoc-Gln (Trt) -OH Fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Leu-OH, fmoc-Tyr (tBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Val-OH, fmoc-Asp (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Thr (tBu) -OH, fmoc-Phe-OH, fmoc-Thr (tBu) -OH, fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-OH and Boc-His (Trt) -OH were washed 3-5 times with solvent to give liraglutide backbone resin.
Further, in the preparation step of the liraglutide main chain resin in S2, the weight ratio of Fmoc-Gly-resin, fmoc-Arg (Pbf) -OH and active agent is 2-4:1-2:0.5-1.5.
The invention also provides a synthesis method of Palmitoyl-Glu-OtBu, which comprises the following steps: weighing H-Glu-OtBu, placing in a container, adding solvent, adding triethylamine at 0-10deg.C, then adding palmitoyl chloride, naturally heating to room temperature, reacting for 2-4 hr, extracting with saturated sodium carbonate, deionized water and saturated saline water sequentially after the reaction is completed, discarding water phase, drying organic phase with anhydrous sodium sulfate, rotary evaporating to obtain oily substance, adding ethyl acetate/petroleum ether mixed solution into oily substance, standing for 2-4 hr, precipitating, recrystallizing, filtering, and vacuum drying to obtain the final product.
According to the synthesis method of Palmitonyl-Glu-OtBu, the molar ratio of H-Glu-OtBu to triethylamine is 1:0.25-0.75.
According to the synthesis method of Palmitoyl-Glu-OtBu, the molar ratio of H-Glu-OtBu to Palmitoyl chloride is 1:1-2.
The invention also provides a synthesis method of the liraglutide intermediate, which comprises the following steps: weighing Palmitoyl-Glu-OtBu, dissolving with a solvent, adding an active intermediate R, adding EDCl under ice bath condition, heating to 20-30 ℃, stirring for reaction for 10-20h, adding EA and water, extracting an organic phase, and evaporating the organic phase to dryness to obtain the liraglutide intermediate.
According to the synthesis method of the liraglutide intermediate, the molar ratio of Palmitoyl-Glu-OtBu to the active intermediate R is 0.8-1:1-2.
According to the synthesis method of the liraglutide intermediate, the weight ratio of Palmitoyl-Glu-OtBu to EDCl is 2-8:1.
The method for synthesizing liraglutide according to the present invention, S3 is advantageousThe preparation method of the pure Lalutide comprises the following steps: under alkaline condition, dissolving liraglutide main chain resin in solvent, adding liraglutide intermediate, coupling reaction between lysine of the liraglutide main chain resin and the liraglutide intermediate, regulating pH to obtain liraglutide resin, adding phenylsilane for 2-5min, and adding Pd (PPh 3 ) 4 And (3) reacting at room temperature for 30-50min, pumping out the reaction solution to remove Fmoc protecting groups, then placing the reaction solution in a cracking solution, reacting for 2-5h at room temperature, filtering, precipitating, centrifuging, washing and drying to obtain liraglutide crude peptide, purifying by adopting a high performance liquid chromatography system, and drying to obtain a pure liraglutide product.
The technical scheme of the invention is further described in detail below with reference to the specific embodiments:
example 1:
a method for synthesizing a liraglutide intermediate, comprising:
weighing 21.5-g H-Glu-OtBu, placing in a three-neck flask, adding 220mL of dichloromethane, adding 6mL of triethylamine at 5 ℃, then adding 34.6g of Palmitoyl chloride, naturally heating to room temperature, reacting for 3 hours, extracting with 120mL of saturated sodium carbonate, 120mL of deionized water and 120mL of saturated saline water for 3 times after the reaction is completed, discarding the water phase, drying the organic phase with anhydrous sodium sulfate, rotary evaporating to remove the solvent to obtain oily matter, adding 250mL of ethyl acetate/petroleum ether mixed solution into the oily matter, placing for 4 hours at 0 ℃, precipitating, recrystallizing, filtering, and vacuum drying to obtain Palmitiyl-Glu-OtBu;
weighing the Palmitonyl-Glu-OtBu, dissolving with DMF, adding active intermediate N-hydroxy-5-norbornene-2, 3-dicarboximide, adding EDCl at 2 ℃, wherein the molar ratio of Palmitonyl-Glu-OtBu to active intermediate R is 1:1.5:0.4, heating to 23 ℃, stirring for reacting for 18h, adding EA and water, wherein the weight of Palmitonyl-Glu-OtBu, EA and water is 1:12:20, extracting an organic phase, evaporating the organic phase to dryness to obtain the liraglutide intermediate with the yield of 81.63% and the purity of 94.28%.
The structural formula is as follows:
；
the determination was carried out using a nuclear magnetic instrument model Bruker AVANCE III HD, deuterated chloroform as solvent, and the determination data were as follows:
1 H NMR (400 MHz，CDCl 3 )：6.25（t，2H，CH）、3.38（t，2H，CH）、1.50-1.75（t，2H，CH 2 ）、3.51（d，2H，CH）、1.98（t，2H，CH 2 ）、1.95（m，2H，CH 2 ）、4.47（m，1H，CH）、8.05（d，1H，NH）、1.40（s，9H，CH 3 ）、2.08（t，2H，CH 2 ）、1.55（m，2H，CH 2 ）、1.31（m，8H，CH 2 ）、1.24（m，14H，CH 2 ）、1.35（m，2H，CH 2 ）、0.86（t，3H，CH 3 ）。
example 2:
a synthetic method of liraglutide intermediate, which is different from example 1: replacing the reactive intermediate with pentafluorophenol;
the structural formula of the liraglutide intermediate is as follows:
；
the nuclear magnetic instrument is adopted for measurement, and the measurement data are as follows:
1 H NMR (400 MHz，CDCl 3 )：1.99（t，2H，CH 2 ）、1.93（m，2H，CH 2 ）、4.48（m，1H，CH）、8.06（d，1H，NH）、1.41（s，9H，CH 3 ）、2.10（t，2H，CH 2 ）、1.57（m，2H，CH 2 ）、1.30（m，8H，CH 2 ）、1.26（m，14H，CH 2 ）、1.38（m，2H，CH 2 ）、0.89（t，3H，CH 3 ）。
example 3:
a synthetic method of liraglutide intermediate, which is different from example 1: replacing the active intermediate with N-hydroxysuccinimide;
the structural formula of the liraglutide active intermediate is as follows:
；
the nuclear magnetic instrument is adopted for measurement, and the measurement data are as follows:
1 H NMR (400 MHz，CDCl 3 )：2.68（t，4H，CH 2 ）、1.96（t，2H，CH 2 ）、1.92（m，2H，CH 2 ）、4.47（m，1H，CH）、8.08（d，1H，NH）、1.43（s，9H，CH 3 ）、2.12（t，2H，CH 2 ）、1.55（m，2H，CH 2 ）、1.29（m，8H，CH 2 ）、1.25（m，14H，CH 2 ）、1.36（m，2H，CH 2 ）、0.91（t，3H，CH 3 ）。
example 4:
a synthetic method of liraglutide intermediate, which is different from example 1: replacing the active intermediate with N-hydroxysulfonic acid succinimide;
the structural formula of the liraglutide intermediate is as follows:
；
the nuclear magnetic instrument is adopted for measurement, and the measurement data are as follows:
1 H NMR (400 MHz，CDCl 3 )：4.26（t，2H，CH 2 ）、3.41（d，2H，CH 2 ）、2.02（s，1H，OH）、1.99（t，2H，CH 2 ）、1.95（m，2H，CH 2 ）、4.51（m，1H，CH）、8.04（d，1H，NH）、1.42（s，9H，CH 3 ）、2.10（t，2H，CH 2 ）、1.52（m，2H，CH 2 ）、1.33（m，8H，CH 2 ）、1.27（m，14H，CH 2 ）、1.39（m，2H，CH 2 ）、0.93（t，3H，CH 3 ）。
example 5:
a method of synthesizing liraglutide, comprising the steps of:
s1: weighing 12g of 2-CTC resin with the substitution degree of 0.8mmol/g, adding the resin into a solid-phase reaction column, washing the resin with DMF for 3 times, swelling the resin with DMF for 35min, dissolving 5.2g of Fmoc-Gly-OH in 20mL of DMF, adding 8.0mL of DIEA under ice bath condition for activation, adding the resin into the solid-phase reaction column for reaction for 1.5h, adding absolute methanol for sealing for 2h, washing the resin with DMF for 4 times, washing the resin with NMP for 3 times respectively, sealing the resin with absolute methanol for 30min, and pumping out the solvent to obtain Fmoc-Gly-resin with the substitution degree of 0.25mmol/g;
s2: weighing 8g Fmoc-Gly-resin, adding the Fmoc-Gly-resin into a solid phase reaction column, washing the Fmoc-Gly-resin for 3 times by using DMF, swelling the Fmoc-Gly-resin by using DMF for 30min, removing Fmoc protection by using 20% DBLK, washing the Fmoc-Gly-resin by using DMF for 2 times and washing the Fmoc-Gly-resin by using NMP for 3 times to obtain H-Gly-resin, dissolving 4.2g Fmoc-Arg (Pbf) -OH, 1.0g HOBt and 0.5g DIC into 50ml of LDMF to form a mixed solution, adding the mixed solution into the solid phase reaction column, reacting the mixed solution for 3H at room temperature, repeating the deprotection step, adding the corresponding amino acid coupling step according to the main chain peptide sequence of liraglutide, fmoc-Gly-OH, fmoc-Arg (Pbf) -OH, fmoc-Val-OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Phe-OH, fmoc-Glu (OtBu) -OH, fmoc-Lys- (Alloc) -OH, fmoc-Ala-OH, fmoc-Gln (Trt) -OH Fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Leu-OH, fmoc-Tyr (tBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Val-OH, fmoc-Asp (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Thr (tBu) -OH, fmoc-Phe-OH, fmoc-Thr (tBu) -OH, fmoc-Gly-OH, fmoc-Glu (OtBu) -OH, fmoc-Ala-OH and Boc-His (Trt) -OH were washed 4 times with DMF and 5 times with DCM, respectively, and dried in vacuo at 55deg.C for 10h to give liraglutide backbone resin;
s3: dissolving 55mg of liraglutide main chain resin in 3.5mL of acetonitrile/water solution, adding the liraglutide intermediate in the embodiment 1, performing coupling reaction on lysine of the liraglutide main chain resin and the liraglutide intermediate, wherein the molar ratio of the liraglutide main chain resin to the liraglutide intermediate is 0.08:1, adjusting the pH value to 10 by using sodium bicarbonate with the concentration of 1mol/L, preparing the liraglutide resin, adding 2.4mL of phenylsilane, reacting for 4min, and then adding 450mg of Pd (PPh 3 ) 4 The reaction is carried out for 40min at room temperature, the reaction solution is pumped out to remove the protecting group, and is washed for 3 times by DMF, then the reaction solution is placed in a lysate (the volume ratio of TFA, phenyl sulfide, anisole and EDT is 90:3:3:4), the reaction is carried out for 3h at room temperature, the resin is filtered out, the filtrate is collected, absolute ethyl ether is added for precipitation, the centrifugation is carried out, the absolute ethyl ether is used for washing the precipitation, the vacuum drying is carried out for 10h at 55 ℃, the crude liraglutide is obtained, the crude liraglutide is dissolved by acetonitrile/water solution, the pure liraglutide is obtained by reversed phase preparation type HPLC system, the wavelength is 215nm, the chromatographic column is C18 filler, and the pure liraglutide is obtained by purification, salt conversion and freeze drying.
Example 6:
a method of synthesizing liraglutide, unlike example 5, in step S3, the liraglutide intermediate in example 1 is replaced with the liraglutide intermediate in example 2.
The other steps were the same as in example 5.
Example 7:
a method of synthesizing liraglutide, unlike example 5, in step S3, the liraglutide intermediate in example 1 is replaced with the liraglutide intermediate in example 3.
The other steps were the same as in example 5.
Example 8:
a method of synthesizing liraglutide, unlike example 5, in step S3, the liraglutide intermediate in example 1 is replaced with the liraglutide intermediate in example 4.
The other steps were the same as in example 5.
Example 9:
unlike example 5, which shows a method for synthesizing liraglutide,
in the step S3, 55mg of the liraglutide main chain resin is dissolved in 4.5mL of DMF, and the liraglutide intermediate in the example 1 is added, so that the coupling reaction is carried out on the lysine of the liraglutide main chain resin and the liraglutide intermediate, wherein the molar ratio of the liraglutide main chain resin to the liraglutide intermediate is 0.05:1, and the pH value is adjusted to 8 by sodium bicarbonate with the concentration of 1mol/L, thus obtaining the liraglutide treeThe fat was reacted for 4min with 2.8mL of phenylsilane, followed by 460mg Pd (PPh) 3 ) 4 The reaction is carried out for 40min at room temperature, the reaction solution is pumped out to remove the protecting group, and is washed for 3 times by DMF, then the reaction solution is placed in a lysate (the volume ratio of TFA, phenyl sulfide, anisole and EDT is 90:3:3:4), the reaction is carried out for 3h at room temperature, the resin is filtered out, the filtrate is collected, absolute ethyl ether is added for precipitation, the centrifugation is carried out, the absolute ethyl ether is used for washing the precipitation, the vacuum drying is carried out for 10h at 55 ℃, the crude liraglutide is obtained, the crude liraglutide is dissolved by acetonitrile/water solution, the pure liraglutide is obtained by reversed phase preparation type HPLC system, the wavelength is 215nm, the chromatographic column is C18 filler, and the pure liraglutide is obtained by purification, salt conversion and freeze drying.
Example 10:
in order to further improve the yield of the synthetic liraglutide, the invention adopts effective measures which also comprise: and (3) replacing part of the phenylsulfide in the cracking liquid in the step (S3) with 4-methyl-5- (2-acetoxyethyl) thiazole, so that the cracking efficiency of the liraglutide resin can be improved, and the yield of the liraglutide is further improved.
Further, the volume ratio of TFA, phenyl sulfide, 4-methyl-5- (2-acetoxyethyl) thiazole, anisole and EDT in the lysate is 85-90:1-3:1-3:1-5:1-5.
Unlike example 5, which shows a method for synthesizing liraglutide,
in step S3, 55mg of the liraglutide main chain resin was dissolved in 3.5mL of acetonitrile/water solution, and the liraglutide intermediate of example 1 was added to perform a coupling reaction of lysine of the above-mentioned liraglutide main chain resin and the liraglutide intermediate, wherein the molar ratio of the liraglutide main chain resin to the liraglutide intermediate was 0.08:1, pH was adjusted to 10 with sodium bicarbonate having a concentration of 1mol/L to prepare a liraglutide resin, 2.4mL of phenylsilane was added to react for 4min, and then 450mg of Pd (PPh 3 ) 4 The reaction is carried out for 40min at room temperature, the reaction solution is pumped out to remove Fmoc protecting groups, and the Fmoc protecting groups are washed for 3 times by DMF, then the Fmoc protecting groups are placed in a lysate (TFA, phenylthiofide, 4-methyl-5- (2-acetoxyethyl) thiazole, anisole and EDT with the volume ratio of 90:2:1:3:4) for reaction for 3h at room temperature, the resin is filtered out, and the filtration is collectedAdding anhydrous diethyl ether into the solution to precipitate, centrifuging, washing the precipitate with anhydrous diethyl ether, vacuum drying at 55deg.C for 10h to obtain crude liraglutide, dissolving with acetonitrile/water solution, purifying with reversed phase preparative HPLC system with wavelength of 215nm and chromatographic column as C18 filler, transferring salt, and lyophilizing to obtain pure liraglutide.
Example 11:
unlike example 5, in step S3, the volume ratio of TFA, phenylsulfide, 4-methyl-5- (2-acetoxyethyl) thiazole, anisole and EDT in the lysate is 90:1:2:3:4.
Comparative example 1:
liraglutide was synthesized according to the experimental method in prior art CN 103980358A.
The experimental results are as follows:
1. yield of crude liraglutide peptide
The yields of the crude liraglutide peptides obtained in examples 5 to 11 and comparative example 1 are shown in Table 1.
TABLE 1 yield of crude liraglutide peptides
As can be seen from Table 1, the yields of the crude liraglutide peptides in examples 5 to 9 are higher than 85% and higher than those of comparative example 1, which shows that the active intermediates are grafted to Palmitonyl-Glu-OH to prepare the crude liraglutide intermediate, and then the liraglutide intermediate is subjected to coupling reaction with lysine of the main chain resin of the liraglutide to prepare the crude liraglutide peptide, so that the yield of the crude liraglutide peptide is improved, and the possible crude liraglutide intermediate has higher grafting activity and can be better grafted to lysine to obtain the crude liraglutide peptide with higher yield.
As can also be seen from Table 1, the yield of the crude liraglutide peptide in examples 10-11 is higher than 90%, and the yields of the crude liraglutide peptide in comparative examples 5 and 10-11 are higher than those of example 5, which indicates that the addition of 4-methyl-5- (2-acetoxyethyl) thiazole to the lysate in step S3 can better cleave the resin to obtain crude liraglutide peptide with higher yield, instead of part of the dimethyl sulfide.
2. Yield and purity of liraglutide pure product
The yields and purities of the pure liraglutide products obtained in examples 5 to 11 and comparative example 1 were measured.
TABLE 2 yield and purity of Liraglutide pure product
As can be seen from Table 2, the yield of the pure liraglutide in examples 5-9 is higher than 75%, the purity is higher than 99%, and the purity is higher than that of comparative example 1, which shows that the active intermediate is grafted to Palmitoly-Glu-OtBu to prepare the liraglutide intermediate, and then the liraglutide intermediate is subjected to coupling reaction with lysine of the liraglutide main chain resin to prepare the crude liraglutide, so that the yield of the crude liraglutide is improved, and the possible liraglutide intermediate has higher grafting activity and can be grafted to lysine better to obtain the pure liraglutide with higher yield and purity. The yield of the pure liraglutide in examples 10-11 is higher than 80%, and the yields of the crude liraglutide in comparative examples 5 and 10-11 are higher than those of example 5, which indicates that the addition of 4-methyl-5- (2-acetoxyethyl) thiazole to the lysate in step S3 can replace part of the phenylthio-ether, so that the resin can be better cracked, the crude liraglutide with higher yield can be obtained, and the pure liraglutide with higher yield can be obtained.
The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.

Claims (7)

1. A method of synthesizing liraglutide, comprising the steps of:
s1: firstly, under the condition of an activating agent, synthesizing Fmoc-Gly-resin by solid phase of the resin and glycine;
s2: deprotection of the Fmoc-Gly-resin, and sequential coupling according to the amino acid sequence of the liraglutide main chain peptide by adopting a solid phase synthesis method to obtain the liraglutide main chain resin;
s3: under alkaline conditions, carrying out coupling reaction on lysine of the liraglutide main chain resin and a liraglutide intermediate, adjusting pH, cracking to obtain a crude product, and purifying to obtain a pure liraglutide;
the preparation method of the liraglutide intermediate comprises the following steps:
weighing H-Glu-OtBu, placing in a container, adding a solvent, adding triethylamine at 0-10 ℃, then adding Palmitoyl chloride, naturally heating to room temperature, reacting for 2-4 hours, extracting with saturated sodium carbonate, deionized water and saturated saline water sequentially after the reaction is completed, discarding a water phase, drying an organic phase with anhydrous sodium sulfate, performing rotary evaporation to obtain oily matter, adding an ethyl acetate/petroleum ether mixed solution into the oily matter, standing for 2-4 hours, precipitating, recrystallizing, filtering, and performing vacuum drying to obtain Palmitiyl-Glu-OtBu;
weighing Palmitoyl-Glu-OtBu, dissolving with a solvent, adding active intermediate N-hydroxy-5-norbornene-2, 3-dicarboximide or pentafluorophenol or N-hydroxysulfonic acid succinimide or N-hydroxysuccinimide, adding EDCl under ice bath condition, heating to 20-30 ℃, stirring for reacting for 10-20h, adding EA and water, extracting an organic phase, and evaporating the organic phase to dryness to obtain liraglutide intermediate;
the cracking solution is a mixture of TFA, phenylthiofide, anisole, 4-methyl-5- (2-acetoxyethyl) thiazole and EDT, and the volume ratio of the cracking solution to the EDT is 85-90:1-3:1-3:1-5:1-5.
2. The method for synthesizing liraglutide according to claim 1, characterized in that: the activator is selected from at least one of HOBt, DIC, DIEA, HATU, DIPEA and PyBOP.
3. The method for synthesizing liraglutide according to claim 1, characterized in that: the alkali used under the alkaline condition is at least one selected from triethylamine, N-methylmorpholine, N-diisopropylethylamine, sodium acetate, potassium acetate, sodium phosphate, disodium hydrogen phosphate, potassium phosphate, dipotassium hydrogen phosphate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate and sodium hydroxide.
4. The method for synthesizing liraglutide according to claim 1, characterized in that: the molar ratio of the liraglutide main chain resin to the liraglutide intermediate is 0.05-0.1:1.
5. The method for synthesizing liraglutide according to claim 1, characterized in that: the temperature of the coupling reaction is-20 ℃ to 60 ℃.
6. The method for synthesizing liraglutide according to claim 1, characterized in that: the pH range is 5-14.
7. The method for synthesizing liraglutide according to claim 1, characterized in that: the molar ratio of Palmitonyl-Glu-OtBu to active intermediate is 0.8-1:1-2.
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